PROJECT 1: SUMMARY HIV infection of the CNS is believed to play an important role in the development of HIV associated neurocognitive disorders (HAND). In our preliminary studies we identified expanded CD163+/CD16+ monoctyes in circulation as well as CD163+/CD16+ macrophages/microglia in CNS in HIV/SIV infection. These cells likely represent an immune polarizing function, contributing to impaired cellular immunity in HIV infection. It is likely that immune polarization of macrophages toward and immune polarized or suppressive phenotype contributes to HIV persistence in the setting of antiretroviral therapy and contributes to the neurocognitive impairment that persists, despite antiviral therapy. In our recent preliminary studies presented here, we identified increased expression of ectonucleotidase enzymes on the surface of macrophages/microglia in CNS in rhesus macaques infected with SIV. We further provide evidence using an inhibitor of CD39 (and ectonucleotidase), showing inhibition of CD16 and CD163 expression on primary monocytes/macrophages. It is likely, therefore, that pharmacologic modulation of ATP hydrolysis can have therapeutic implications for restoring antiviral immune responses in HIV infection. These observations, together with the immunosuppressive and immune polarizing role of these enzymes, suggest that successful eradication of reservoirs of HIV infection in the CNS will require targeted approaches to prevent increased ATP hydrolysis in order to accomplish eradication of reservoirs of HIV infection, particularly in the CNS. Here we propose in vivo and ex vivo using the SIV infected rhesus macaque model in the context of the following Specific Aims: Specfic Aim 1: To test the hypothesis that Adenosine production, as a consequence of ATP hydrolysis, modulates monocyte maturation (i.e. CD16 gene expression), M2 polarization, and SIV specific immune responses in vitro. Here we will determine the ability of antagonists of CD39 (POM-1) and CD73 (APCP) enzyme activity to inhibit CD16 expression on monocytes in response to treatment with Giant Cell Tumor Conditioned Medium or Macrophage Colony-Stimulating Factor (M-CSF), as inducers of M2 maturation and CD16 expression. In our preliminary data we present evidence suggesting the ability of POM- 1 treatment to inhibit the induction of CD16 expression on monocytes in vitro. As this effect is likely mediated by preventing adenosine formation, we will further investigate the ability of adenosine agonists to promote the maturation of non-classical monocyte subsets (CD16+/CD163+) monocytes derived from normal blood donors and uninfected rhesus macaques. Our in vitro studies will determine the ability of CD39, CD73, and adenosine receptor (ie. Adora2b) antagonists to reduce the maturation of CD16+ monocytes in response to M-CSF treatment in vitro, and further, for the ability to reduce CD16 gene expression on cells previously differentiated with M-CSF. The ability of compounds to inhibit macrophage maturation (ie CD16 expression), M2 polarization, and to augment T cell responses will be tested alone and in combination with pharmacologic strategies in the other two projects: cFMS tyrosine kinase inhibitors (Project 2) and NK-1R specifc antagonist (aprepitant) (Project 3). Specific Aim 2: To test the hypothesis that pharmacologic intervention with a lead compound targeting adenosine metabolism (CD39, CD73 or Ador2B antagonists), alone and in combination with tyrosine kinase inhibitors and/or aprepitant, will restore normal monocyte/macrophage dynamics and phenotype, virus specific immune responses in SIV infected rhesus macaques in the presence and absence of cART therapy, and reduce CNS and lymphoid viral reservoirs. In our preliminary studies we demonstrate increased production of CD16+/BrdU positive monocytes in SIV infected animals the periphery at 3 days post BrdU administration. Further, we observe increased T cell clearance in SIV infected animals and importantly a correlation between the rate of T cell turnover and the rate of production of CD16+/BrdU+ positive cells. In our studies performed in this Aim we will select the best drug candidate (lead compound) based on in vitro studies performed in Aim 1, information regarding bioavailability, toxicity, and its potential to synergize with compounds developed in Projects 2 and 3. Here we will investigate the ability of pharmacologic intervention with our lead compound to normalize CD16+/BrdU monocyte and T cell turnover in vivo in SIV infected macaques treated with this compound alone, or in the presence of cART treatment. These studies targeting ATP metabolism to address the persistence of HIV infected reservoirs in the CNS should provide a framework for therapeutic/elimination strategies based on restoration of immune responses, and the interaction of this and other strategies targeting M2 polarization.